Methods for selectively suppressing non-target sequences

ABSTRACT

The invention generally relates to negative selection of nucleic acids. The invention provides methods and systems that remove unwanted segments of nucleic acid in a sample so that a target gene or region of interest may be analyzed without interference from the unwanted segments. A sample is obtained that includes single-stranded nucleic acid with one or more unwanted segments. Complementary nucleic acid is added to the single-stranded nucleic acid to create a double-stranded region that includes the unwanted segment. The double-stranded region is then digested, leaving single-stranded nucleic acid that includes the target gene or region of interest. This allows paralogs, pseudogenes, repetitive elements, and other segments of the genome that may be similar to the target gene or region of interest to be removed from the sample.

TECHNICAL FIELD

The invention generally relates to negative selection of nucleic acids.

BACKGROUND

The advent of high-throughput DNA sequencing has the potential torevolutionize modern biology and transform diagnostic medicine.Instruments for next-generation sequencing (NGS) continue to generatemore data and become more inexpensive at a rate far outpacing Moore'sLaw. However, the most popular sequencers have an extremely short readlength, limiting their ability to characterize any gene containingparalogous sequence or repetitive elements. As nearly two thirds of thegenome is highly repetitive and over 20,000 pseudogenic regions exist,much of the genome is very difficult to characterize in a modernwhole-genome sequencing experiment. Unfortunately, for many genes ofclinical interest, characterizing those genes is made difficult by thepresence of paralogs, pseudogenic homologs, and other segments of thegenome that may be similar to the gene of interest and thus stymieattempts to detect, sequence, or isolate the gene of interest. As aresult, despite the power of NGS instruments, some disease-related genesand mutations, even where known, are difficult to detect.

SUMMARY

The invention provides methods and systems that remove unwanted segmentsof nucleic acid in a sample so that a target gene or region of interestmay be analyzed without interference from the unwanted segments. Asample is obtained that includes single-stranded nucleic acid with oneor more unwanted segments. Primers that are specific or preferentiallybind to the unwanted segment are hybridized to the single-strandednucleic acid within the unwanted region or in a non-repetitive sectionupstream of the unwanted region and extended by a polymerase to create adouble-stranded region that includes the unwanted segment. Thedouble-stranded region is then digested, leaving single-stranded nucleicacid that includes the target gene or region of interest. This allowsparalogs, pseudogenes, repetitive elements, and other segments of thegenome that may be similar to the target gene or region of interest tobe removed from the sample. The target gene or region of interest maythus be detected or characterized by analysis without interference fromthe unwanted segments. This may provide an improved ability to detectfeatures such as disease-related genes and mutations, thus improving theclinical value of NGS technologies.

Systems and methods of the invention may be used to remove unwantedregions from genomic DNA (such as homologous genes, pseudogenes, orrepetitive elements) prior to any DNA-based experimental procedure,including but not limited to microarray hybridization, quantitative orstandard polymerase chain reaction, multiplex target capture, or DNAsequencing (either targeted or shotgun). Systems and methods of theinvention provide for the identification of mutations in previouslydifficult-to-characterize genes, and therefore allow practitioners toexpand the number of genes included in a targeted or whole-genomesequencing assay.

In certain aspects, the invention provides a method of removing unwantedsegments of a nucleic acid from a sample. The method includes annealinga nucleic acid primer to a portion of a single-stranded nucleic acidthat flanks an unwanted segment of the nucleic acid, extending theannealed primer in order to create a double-stranded region thatincludes the unwanted segment; and digesting the double-stranded region,thereby removing the unwanted segment from the nucleic acid.

The nucleic acid in the sample may include DNA, RNA, modified nucleicacids, or combinations thereof. The method may include obtaining asample from a subject and denaturing double-stranded DNA in the sample.Denaturing can include the use of methods such as exposing the sample toheat, a detergent, or an acidic or basic solution.

The primer may be annealed within the unwanted segment or within an areaupstream of the unwanted segment and extended. A pair or a number ofprimers may be used and primers that flank the unwanted segment may beused. In certain embodiments, a plurality of primers are annealed to aplurality of portions of that nucleic acid that flank an unwantedsegment. The primer or primers are preferably extended using apolymerase enzyme under conditions sufficient to cause extension of theprimer in a template-dependent manner. In some embodiments, a primer oroligonucleotide is hybridized to the unwanted segment to create thedouble-stranded region containing the unwanted segment without need foran extension step.

The double-stranded region is digested. This can include exposing thesample to an enzyme that preferentially digests double-stranded nucleicacid such as certain double-stranded endonucleases, restrictionendonucleases, or nicking enzymes. After digestion, the enzyme may bede-activated (e.g., by heat, chemicals, etc.). Digestion preferablyresults in intact genomic DNA lacking one or more unwanted segment andthat is compatible with a nucleic acid analysis assay. Nucleic acid thatis not digested may be analyzed by a nucleic acid analysis assay. Assayssuitable for analysis of the remaining un-digested nucleic acid may makeuse of molecular inversion probe capture, hybrid capture, Haloplex,sequencing (e.g., Sanger sequencing, NGS, or both), other methodologies,or combinations thereof. Where the unwanted segment is a paralog, apseudogene, or non-paralogous repetitive element, such elements may beremoved from the sample by methods of the invention.

In certain aspects, the invention provides a method of removing nucleicacid from a sample. The method includes annealing at least oneoligonucleotide to single-stranded DNA in a sample, wherein thesingle-stranded DNA comprises target and non-target sequence. Theoligonucleotide may be annealed to the non-target sequence to createdouble-stranded DNA that includes the non-target sequence or theoligonucleotide may be annealed elsewhere and extended to createdouble-stranded DNA that includes the non-target sequence. Thenon-target sequence is removed from the sample by digesting thedouble-stranded DNA. The target sequence may be analyzed using, e.g.,molecular inversion probes, microarray hybridization, multiplexligation-dependent probe amplification (MLPA), sequencing,fingerprinting techniques such as RFLP/AFLP, chromatography, others, orcombinations thereof. In some embodiments, the method includes firstobtaining the sample from a subject and denaturing double-strandedsubject DNA to produce the single-stranded DNA. Preferably, thatsingle-stranded DNA consists essentially of genomic DNA from the subjectprior to the annealing of the oligo. The annealing may include annealinga pair of oligonucleotides to the single-stranded DNA at sites thatflank the non-target sequence (i.e., to remove both strands of theunwanted segment or non-target sequence. In certain embodiments, thetarget and non-target sequence are both located on at least one singlestrand of the single-stranded DNA, and extending the at least oneoligonucleotide and digesting the double-stranded DNA results inremoving the non-target sequence from the at least one single strand ofthe single-stranded DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrams a method of removing unwanted segments of a nucleicacid.

FIG. 2 illustrates methods according to certain embodiments.

FIG. 3 gives a diagram of a system according to embodiments of theinvention.

DETAILED DESCRIPTION

To enable the characterization of difficult genomic regions usinghigh-throughput short-read sequencing, the invention provides methodsfor the removal of unwanted genomic regions from a population of DNAmolecules (e.g. genomic DNA). Most DNA-based techniques rely on theamplification of specific regions of interest or sequencing librarymolecules in a positive selection process (e.g. amplification utilizingprimers that are unique to a single paralog). Methods of the inventioninstead involve a negative selection technique that removes anyundesired analogous sequence, allowing application of standardhigh-throughput sequencing techniques or other analyses to anydifficult-to-characterize gene of interest.

Applicability of methods of the invention may be illustrated byreference to two exemplary genes of interest for which directhigh-throughput sequencing-based approaches are currently insufficient.One gene is “glucosidase beta acid,” or GBA, which has been implicatedas causative in Gaucher disease. Currently, long-range polymerase chainreaction experiments are required to characterize this gene, as apseudogene with nearly identical sequence exists a mere 15,000 basepairs away. By removing this pseudogenic region using the invention, GBAcan be characterized with high specificity, enabling construction of agenetic screen for Gaucher disease. This gene is a suitable target formethods of the invention, as it is relatively small and contains nearbyunique flanking sequence.

An additional gene of interest is “survival of motor neuron 1,” or SMN1.This gene has been implicated in spinal muscular atrophy. Currently, dueto the presence of a paralogous gene known as SMN2 that is 100,000 basepairs away from SMN1, characterization of SMN1 is extremely challenging.By removing SMN2 using the invention, SMA could be screened for with ahigh-throughput sequencing approach that would not require a complexstatistical model.

Additionally, novel causative mutations in genes such as SMN1 could alsobe identified. This gene is a suitable target for methods of theinvention. It is of a suitable size and flanked by highly repetitiveregions.

FIG. 1 diagrams a method 101 of removing unwanted segments of a nucleicacid from a sample according to embodiments of the invention. The methodincludes obtaining 105 a sample that includes nucleic acid. Anoligonucleotide is annealed 109 to an unwanted segment of the nucleicacid or a portion of the nucleic acid that flanks an unwanted segment ofthe nucleic acid. In embodiments in which the oligonucleotide flanks theunwanted segment, the oligonucleotide is extended 113 to create adouble-stranded region that includes the unwanted segment. Inembodiments in which the oligonucleotide is annealed to the unwantedsegment, a double-stranded region that includes the unwanted segment iscreated by virtue of the hybridization of the oligonucleotide at thatsegment. The double-stranded region is digested 117, thus removing theunwanted segment from the nucleic acid. This allows for a region or geneof interest to be analyzed 121.

The sample that includes nucleic acid may be obtained 105 by anysuitable method. The sample may be obtained from a tissue or body fluidthat is obtained in any clinically acceptable manner. Body fluids mayinclude mucous, blood, plasma, serum, serum derivatives, bile, blood,maternal blood, phlegm, saliva, sweat, amniotic fluid, menstrual fluid,mammary fluid, follicular fluid of the ovary, fallopian tube fluid,peritoneal fluid, urine, and cerebrospinal fluid (CSF), such as lumbaror ventricular CSF. A sample may also be a fine needle aspirate orbiopsied tissue. A sample also may be media containing cells orbiological material. Samples may also be obtained from the environment(e.g., air, agricultural, water and soil) or may include researchsamples (e.g., products of a nucleic acid amplification reaction, orpurified genomic DNA, RNA, proteins, etc.).

Isolation, extraction or derivation of genomic nucleic acids may beperformed by methods known in the art. Isolating nucleic acid from abiological sample generally includes treating a biological sample insuch a manner that genomic nucleic acids present in the sample areextracted and made available for analysis. Generally, nucleic acids areextracted using techniques such as those described in Green & Sambrook,2012, Molecular Cloning: A Laboratory Manual 4 edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2028 pages), thecontents of which are incorporated by reference herein. A kit may beused to extract DNA from tissues and bodily fluids and certain such kitsare commercially available from, for example, BD Biosciences Clontech(Palo Alto, Calif.), Epicentre Technologies (Madison, Wis.), GentraSystems, Inc. (Minneapolis, Minn.), and Qiagen Inc. (Valencia, Calif.).User guides that describe protocols are usually included in such kits.

It may be preferable to lyse cells to isolate genomic nucleic acid.Cellular extracts can be subjected to other steps to drive nucleic acidisolation toward completion by, e.g., differential precipitation, columnchromatography, extraction with organic solvents, filtration,centrifugation, others, or any combination thereof. The genomic nucleicacid may be resuspended in a solution or buffer such as water, Trisbuffers, or other buffers. In certain embodiments the genomic nucleicacid can be re-suspended in Qiagen DNA hydration solution, or otherTris-based buffer of a pH of around 7.5.

Any nucleic acid may be analyzed using methods of the invention. Nucleicacids suitable for use in aspects of the invention may include withoutlimit genomic DNA, genomic RNA, synthesized nucleic acids, whole orpartial genome amplification product, and high molecular weight nucleicacids, e.g. individual chromosomes. In certain embodiments, a sample isobtained that includes double-stranded DNA, such as bulk genomic DNAfrom a subject, and the double-stranded DNA is then denatured.

Double stranded nucleic acid may be denatured using any suitable methodsuch as, for example, through the use of heat, detergent incubation, oran acidic or basic solution.

FIG. 2 illustrates the progress of methods according to certainembodiments. As shown in FIG. 2, methods may start with double strandedDNA (dotted shading if not otherwise hatched) that contains a gene ofinterest (first angled hatching pattern) and a paralog of the gene ofinterest (second angled hatching pattern). It will be appreciated thatmethods of the invention may operate starting with any suitable nucleicacid such as double- or single-stranded DNA or RNA or any combinationthereof. The unwanted segment may be any sequence for which removal isdesired from the starting nucleic acid. For example, the unwantedsegment may include a paralog or homolog of a gene or region ofinterest; a pseudogene; or non-paralogous repetitive element. As usedherein, homolog refers to a gene related to a second gene by descentfrom a common ancestral DNA sequence. Homolog describes the relationshipbetween genes separated by the event of speciation (i.e., orthology) orto the relationship between genes separated by the event of geneticduplication (i.e., paralogy). Orthologs generally refers to genes indifferent species that evolved from a common ancestral gene byspeciation. Normally, orthologs retain the same function in the courseof evolution and paralogs are genes related by duplication within agenome. See Fitch, 1970, Distinguishing homologs from analogousproteins, Syst Biol 19(2):99-113 and Jensen, 2001, Orthologs andparalogs—we need to get it right, Genome Biol 2(8):1002-1002.3.Pseudogenes include dysfunctional relatives of genes that have losttheir protein-coding ability or are otherwise no longer expressed in thecell. Methods of the invention may be used to target a pseudogene thatis present as a homolog to another gene or pseudogene within a sampleand methods of the invention may be used to target a pseudogene that ispresent even where no known homologs of the pseudogene are suspected toalso be present in the sample.

As illustrated in FIG. 2, the double-stranded DNA is denatured into itstwo complementary strands prior to primer hybridization. Any suitablemethod may be used to denature nucleic acid. Heat-based denaturing is aprocess by which double-stranded nucleic acid unwinds and separates intosingle-stranded strands. Heat denaturation of a nucleic acid of anunknown sequence typically uses a temperature high enough to ensuredenaturation of even nucleic acids having a very high GC content, e.g.,95° C.−98° C. in the absence of any chemical denaturant. It is wellwithin the abilities of one of ordinary skill in the art to optimize theconditions (e.g., time, temperature, etc.) for denaturation of thenucleic acid. Temperatures significantly lower than 95° C. can also beused if the DNA contains nicks (and therefore sticky overhangs of lowTm), sequence of sufficiently low Tm, or chemical additives such asbetaine.

Denaturing nucleic acids with the use of pH is also well known in theart, and such denaturation can be accomplished using any method known inthe art such as introducing a nucleic acid to high or low pH, low ionicstrength, and/or heat, which disrupts base-pairing causing adouble-stranded helix to dissociate into single strands. For methods ofpH-based denaturation see, for example, Ageno et al., 1969, The alkalinedenaturation of DNA, Biophys J 9:1281-1311.

Nucleic acids can also be denatured via electro-chemical means, forexample, by applying a voltage to a nucleic acid within a solution bymeans of an electrode. Varying methods of denaturing by applying avoltage are discussed in detail in U.S. Pat. Nos. 6,197,508; 5,993,611.After denaturation, unwanted segments can be targeted for removal.

Methods of the invention include targeting unwanted segments of nucleicacid for removal. An unwanted segment of nucleic acid can be targetedfor removal by making it into a double-stranded segment. The unwantedsegment can be made double-stranded by hybridizing a complementaryoligonucleotide to the unwanted segment, by hybridizing a complementaryoligonucleotide to a genomic segment flanking the unwanted segment andextending the oligonucleotide, or a combination thereof (e.g., anoligonucleotide can be hybridized so that it sits partially within theunwanted segment and then extended via methods described herein).

In certain embodiments, the oligonucleotide to be hybridized is a primerthat is unique to the unwanted segment. For example, methods may includeusing a primer that is unique to a certain paralog or other element. Theinvention provides methods of making a primer and primer extensionreactions that are unique to a paralog or similar segment by includingor using a primer with a 3′ end that terminates on a differentiatingbase (i.e., the 3′-most base or bases of the primer may be complementaryto a base or bases that appear only in association with the segment(e.g., paralog) targeted for removal.

In some embodiments, double stranded DNA is created by hybridizationalone (e.g., rather than by using oligonucleotide primer with polymeraseextension). One or more long segments of nucleic acid complementary tothe unwanted segments could be used. For example, long segments ofsynthetic DNA could be used. The segments of complementary nucleic acidcould have any suitable length such as, for example, tens of bases,hundreds of bases, length of an exon, length of a gene, etc. Use of oneor more long segments of nucleic acid complementary to the unwantedsegments (e.g., followed by digestion of dsDNA) may provide forenrichment of, for example, target relative to non-target.

As noted above, the recognition site for the oligonucleotide, primer, orcomplementary nucleic acid may flank the unwanted segment, lie withinthe unwanted segment, or both. Additionally, methods may include usingone or any suitable number of oligonucleotides or primers to target anunwanted segment or segments of nucleic acid.

In the non-limiting, illustrative embodiment shown in FIG. 2, primers(cross-hatching pattern) are annealed to unique genomic segmentsflanking the paralogous region. The primer may be annealed at anysuitable location. For example, it may be preferable to anneal any ofthe one or more primers to a portion within 50 or fewer bases from theunwanted segment, although it may not be necessary to anneal the primerswithin 50 bases of the unwanted region. As shown in FIG. 2, primers areannealed at locations that flank the unwanted segment, i.e., each primerof a pair hybridizes to its target strand in a region that flanks the 5′end of the unwanted segment. In this way, extension of the primers willresult in most or all of the unwanted segments being present inexclusively double-stranded form, whereas the desired region(s) shouldremain in a primarily single-stranded state.

In certain embodiments, polymerase (drawn as an open circle in FIG. 2)is used to perform second-strand synthesis over the paralogous region.Extending the annealed primer creates a double-stranded region thatincludes the unwanted segment. The primer is extended using a polymeraseenzyme under conditions sufficient to cause extension of the primer in atemplate-dependent manner. Suitable polymerase enzymes include phi29,Bst, Exo-minus E. Coli Polymerase I, Taq Polymerase, and T7 PolymeraseI.

An enzymatic digestion (the digestion enzyme is represented by adarkened hexagon in FIG. 2) is then used to degrade only thedouble-stranded paralogous region, leaving behind the gene of interest.Any suitable digestion platform may be employed such as, for example,dsDNAse, fragmentase, a non-specific nicking enzyme such as a modifiedVvn, restriction enzymes such as MspJI and FspEI, and a combination ofUSER plus T7 endonuclease I. Thermo Scientific dsDNase is an engineeredshrimp DNase designed for rapid and safe removal of contaminatinggenomic DNA from RNA samples. It is an endonuclease that cleavesphosphodiester bonds in DNA to yield oligonucleotides with 5′-phosphateand 3′-hydroxyl termini. Highly specific activity towardsdouble-stranded DNA ensures that RNA and single-stranded DNA such ascDNA and primers are not cleaved. dsDNase is easily inactivated bymoderate heat treatment (55° C.). Thermo Scientific dsDNAse is availablefrom Thermo Fisher Scientific, Inc. (Waltham, Mass.).

Fragmentase includes the enzyme sold under the trademark NEBNEXT dsDNAfragmentase by New England Biolabs (Ipswich, Mass.). NEBNEXT dsDNAfragmentase generates dsDNA breaks in a time-dependent manner to yield50-1,000 bp DNA fragments depending on reaction time. NEBNext dsDNAFragmentase contains two enzymes, one randomly generates nicks on dsDNAand the other recognizes the nicked site and cuts the opposite DNAstrand across from the nick, producing dsDNA breaks. The resulting DNAfragments contain short overhangs, 5″-phosphates, and 3″-hydroxylgroups. The random nicking activity of NEBNext dsDNA Fragmentase hasbeen confirmed by preparing libraries for next-generation sequencing. Acomparison of the sequencing results between genomic DNA (gDNA) preparedwith NEBNext dsDNA fragmentase and with mechanical shearing demonstratesthat the NEBNext dsDNA Fragmentase does not introduce any detectablebias during the sequencing library preparation and no difference insequence coverage is observed using the two methods

The Vibrio vulnificus nuclease, Vvn, is a non-specific periplasmicnuclease capable of digesting DNA and RNA. It has been suggested thatVvn hydrolyzes DNA by a general single-metal ion mechanism. See Li, etal., 2003, DNA binding and cleavage by the periplasmic nuclease Vvn: anovel structure with a known active site, EMBO J 22(15):4014-4025.

MspJI is a modification dependent endonuclease that recognizes certainmethylation patterns. The most common epigenetic modifications found ineukaryotic organisms are methylation marks at CpG or CHG sites. A subsetof these modified sites are recognized and cleaved by MspJI. MspJI isavailable from New England Biolabs. T7 Endonuclease I recognizes andcleaves non-perfectly matched DNA, cruciform DNA structures, Hollidaystructures or junctions, hetero-duplex DNA and more slowly, nickeddouble-stranded DNA. The cleavage site is at the first, second or thirdphosphodiester bond that is 5′ to the mismatch. The protein is theproduct of T7 gene 3. Any other suitable enzyme for digesting the targetunwanted segments may be used.

The added enzymes may then be deactivated using an irreversible heat orchemical treatment, leaving genomic DNA lacking an intact undesiredregion(s) yet still compatible with any downstream assay (e.g. molecularinversion probe capture or any other library construction methodology).

The digesting step results in intact genomic DNA lacking one or moreunwanted segment and that is compatible with a nucleic acid analysisassay. This DNA can then be utilized for any downstream assay.Downstream assays may include molecular inversion capture, sequencing,others, or a combination thereof.

Methods of the invention can be used to negatively select outpseudogenic regions from the genome. Methods of the invention can becombined with a genetic test, screening, or other assay in order toscreen patients for mutations in a gene (e.g., GBA, SMN1, or other genescontaining paralogous regions). Some background may be found inpublished international patent application WO 2013/191775, to NugenTechnologies, Inc.

After removing the unwanted segment from the nucleic acid, the samplemay be enriched for genes of interest using methods known in the art,such as hybrid capture. Methods suitable for use may be found discussedin U.S. Pat. Nos. 8,529,744; 7,985,716; 7,666,593; and 6,613,516. Aswill be described in more detail below, a preferable capture method usesmolecular inversion probes.

Nucleic acids, including genomic nucleic acids, can be fragmented usingany of a variety of methods, such as mechanical fragmenting, chemicalfragmenting, and enzymatic fragmenting. Methods of nucleic acidfragmentation are known in the art and include, but are not limited to,DNase digestion, sonication, mechanical shearing, and the like. U.S. Pub2005/0112590 provides a general overview of various methods offragmenting known in the art.

Genomic nucleic acids can be fragmented into uniform fragments orrandomly fragmented. In certain aspects, nucleic acids are fragmented toform fragments having a fragment length of about 5 kilobases or 100kilobases. Desired fragment length and ranges of fragment lengths can beadjusted depending on the type of nucleic acid targets one seeks tocapture and the design and type of probes such as molecular inversionprobes (MIPs) that will be used. Chemical fragmentation of genomicnucleic acids can be achieved using methods such as a hydrolysisreaction or by altering temperature or pH. Nucleic acid may befragmented by heating a nucleic acid immersed in a buffer system at acertain temperature for a certain period to time to initiate hydrolysisand thus fragment the nucleic acid. The pH of the buffer system,duration of heating, and temperature can be varied to achieve a desiredfragmentation of the nucleic acid. Mechanical shearing of nucleic acidsinto fragments can be used e.g., by hydro-shearing, trituration througha needle, and sonication. The nucleic acid can also be sheared vianebulization, hydro-shearing, sonication, or others. See U.S. Pat. Nos.6,719,449; 6,948,843; and 6,235,501.

Nucleic acid may be fragmented enzymatically. Enzymatic fragmenting,also known as enzymatic cleavage, cuts nucleic acids into fragmentsusing enzymes, such as endonucleases, exonucleases, ribozymes, andDNAzymes. Varying enzymatic fragmenting techniques are well-known in theart. Additionally, DNA may be denatured again as needed after thedigestion and any other sample prep steps. For example, during afragmentation step, ssDNA may anneal to form dsDNA and it may bedesirable to again denature the dsDNA. In certain embodiments, thesample nucleic acid is captured or targeted using any suitable capturemethod or assay such as hybridization capture or capture by probes suchas MIPs.

MIPs, or molecular inversion probes, can be used to detect or amplifyparticular nucleic acid sequences in complex mixtures. Use of molecularinversion probes has been demonstrated for detection of singlenucleotide polymorphisms (Hardenbol et al., 2005, Highly multiplexedmolecular inversion probe genotyping: over 10,000 targeted SNPsgenotyped in a single tube assay, Genome Res 15:269-75) and forpreparative amplification of large sets of exons (Porreca et al., 2007,Multiplex amplification of large sets of human exons, Nat Methods4:931-6, Krishnakumar et al., 2008, A comprehensive assay for targetedmultiplex amplification of human DNA sequences, PNAS 105:9296-301). Oneof the main benefits of the method is in its capacity for a high degreeof multiplexing, because generally thousands of targets may be capturedin a single reaction containing thousands of probes.

In certain embodiments, molecular inversion probes include a universalportion flanked by two unique targeting arms. The targeting arms aredesigned to hybridize immediately upstream and downstream of a specifictarget sequence located on a genomic nucleic acid fragment. Themolecular inversion probes are introduced to nucleic acid fragments toperform capture of target sequences located on the fragments. Accordingto the invention, fragmenting aids in capture of target nucleic acid bymolecular inversion probes. As described in greater detail herein, aftercapture of the target sequence (e.g., locus) of interest, the capturedtarget may further be subjected to an enzymatic gap-filling and ligationstep, such that a copy of the target sequence is incorporated into acircle. Capture efficiency of the MIP to the target sequence on thenucleic acid fragment can be improved by lengthening the hybridizationand gap-filing incubation periods. (See, e.g., Turner et al., 2009,Massively parallel exon capture and library-free resequencing across 16genomes, Nature Methods 6:315-316.) A library of molecular inversionprobes may be created and used in capturing DNA of genomic regions ofinterests (e.g., SMN1, SMN2, control DNA). The library includes aplurality of oligonucleotide probes capable of capturing one or moregenomic regions of interest (e.g., SMN1, SMN2 and control loci) withinthe samples to be tested.

The result of MIP capture as described above is a library of circulartarget probes, which then can be processed in a variety of ways.Adaptors for sequencing may be attached during common linker-mediatedPCR, resulting in a library with non-random, fixed starting points forsequencing. For preparation of a shotgun library, a commonlinker-mediated PCR is performed on the circle target probes, and thepost-capture amplicons are linearly concatenated, sheared, and attachedto adaptors for sequencing. Methods for shearing the linear concatenatedcaptured targets can include any of the methods disclosed forfragmenting nucleic acids discussed above. In certain aspects,performing a hydrolysis reaction on the captured amplicons in thepresence of heat is the desired method of shearing for libraryproduction.

In some embodiments, the amount of target nucleic acid and probe usedfor each reaction is normalized to avoid any observed differences beingcaused by differences in concentrations or ratios. In some embodiments,in order to normalize genomic DNA and probe, the genomic DNAconcentration is read using a standard spectrophotometer or byfluorescence (e.g., using a fluorescent intercalating dye). The probeconcentration may be determined experimentally or using informationspecified by the probe manufacturer.

Similarly, once a locus has been captured, it may be amplified and/orsequenced in a reaction involving one or more primers. The amount ofprimer added for each reaction can range from 0.1 pmol to 1 nmol, 0.15pmol to 1.5 nmol (for example around 1.5 pmol). However, other amounts(e.g., lower, higher, or intermediate amounts) may be used.

A targeting arm may be designed to hybridize (e.g., be complementary) toeither strand of a genetic locus of interest if the nucleic acid beinganalyzed is DNA (e.g., genomic DNA). For MIP probes, whichever strand isselected for one targeting arm will be used for the other one. In thecontext of RNA analysis, a targeting arm should be designed to hybridizeto the transcribed RNA. It also should be appreciated that MIP probesreferred to herein as “capturing” a target sequence are actuallycapturing it by template-based synthesis rather than by capturing theactual target molecule (other than for example in the initial stage whenthe arms hybridize to it or in the sense that the target molecule canremain bound to the extended MIP product until it is denatured orotherwise removed).

A targeting arm may include a sequence that is complementary to oneallele or mutation (e.g., a SNP or other polymorphism, a mutation, etc.)so that the probe will preferentially hybridize (and capture) targetnucleic acids having that allele or mutation. Sequence tags (alsoreferred to as barcodes) may be designed to be unique in that they donot appear at other positions within a probe or a family of probes andthey also do not appear within the sequences being targeted. Uniformityand reproducibility can be increased by designing multiple probes pertarget, such that each base in the target is captured by more than oneprobe.

The length of a capture molecule on a nucleic acid fragment (e.g., atarget nucleic acid or sub-region thereof) may be selected based uponmultiple considerations. For example, where analysis of a targetinvolves sequencing, e.g., with a next-generation sequencer, the targetlength should typically match the sequencing read-length so that shotgunlibrary construction is not necessary. However, it should be appreciatedthat captured nucleic acids may be sequenced using any suitablesequencing technique as aspects of the invention are not limited in thisrespect.

It is also to be appreciated that some target nucleic acids on a nucleicacid fragment are too large to be captured with one probe. Consequently,it may be helpful to capture multiple sub-regions of a target nucleicacid in order to analyze the full target.

Methods of the invention also provide for combining the method offragmenting the nucleic acid prior to capture with other MIP capturetechniques that are designed to increase target uniformity,reproducibility, and specificity. Other MIP capture techniques are shownin U.S. Pub. 2012/0165202, incorporated by reference.

Multiple probes, e.g., MIPs, can be used to amplify each target nucleicacid. In some embodiments, the set of probes for a given target can bedesigned to ‘tile’ across the target, capturing the target as a seriesof shorter sub targets. In some embodiments, where a set of probes for agiven target is designed to ‘tile’ across the target, some probes in theset capture flanking non-target sequence). Alternately, the set can bedesigned to ‘stagger’ the exact positions of the hybridization regionsflanking the target, capturing the full target (and in some casescapturing flanking non-target sequence) with multiple probes havingdifferent targeting arms, obviating the need for tiling. The particularapproach chosen will depend on the nature of the target set. Forexample, if small regions are to be captured, a staggered-end approachmight be appropriate, whereas if longer regions are desired, tilingmight be chosen. In all cases, the amount of bias-tolerance for probestargeting pathological loci can be adjusted by changing the number ofdifferent MIPs used to capture a given molecule.

Probes for MIP capture reactions may be synthesized on programmablemicroarrays because of the large number of sequences required. Becauseof the low synthesis yields of these methods, a subsequent amplificationstep is required to produce sufficient probe for the MIP amplificationreaction. The combination of multiplex oligonucleotide synthesis andpooled amplification results in uneven synthesis error rates andrepresentational biases. By synthesizing multiple probes for eachtarget, variation from these sources may be averaged out because not allprobes for a given target will have the same error rates and biases.

Using methods described herein, a single copy of a specific targetnucleic acid may be amplified to a level that can be sequenced. Further,the amplified segments created by an amplification process such as PCRmay be, themselves, efficient templates for subsequent PCRamplifications.

Amplification or sequencing adapters or barcodes, or a combinationthereof, may be attached to the fragmented nucleic acid. Such moleculesmay be commercially obtained, such as from Integrated DNA Technologies(Coralville, Iowa). In certain embodiments, such sequences are attachedto the template nucleic acid molecule with an enzyme such as a ligase.Suitable ligases include T4 DNA ligase and T4 RNA ligase, availablecommercially from New England Biolabs (Ipswich, Mass.). The ligation maybe blunt ended or via use of complementary overhanging ends. In certainembodiments, following fragmentation, the ends of the fragments may berepaired, trimmed (e.g. using an exonuclease), or filled (e.g., using apolymerase and dNTPs) to form blunt ends. In some embodiments, endrepair is performed to generate blunt end 5′ phosphorylated nucleic acidends using commercial kits, such as those available from EpicentreBiotechnologies (Madison, Wis.). Upon generating blunt ends, the endsmay be treated with a polymerase and dATP to form a template independentaddition to the 3′-end and the 5′-end of the fragments, thus producing asingle A overhanging. This single A can guide ligation of fragments witha single T overhanging from the 5′-end in a method referred to as T-Acloning. Alternatively, because the possible combination of overhangsleft by the restriction enzymes are known after a restriction digestion,the ends may be left as-is, i.e., ragged ends. In certain embodimentsdouble stranded oligonucleotides with complementary overhanging ends areused.

In certain embodiments, one or more bar code is attached to each, any,or all of the fragments. A bar code sequence generally includes certainfeatures that make the sequence useful in sequencing reactions. The barcode sequences are designed such that each sequence is correlated to aparticular portion of nucleic acid, allowing sequence reads to becorrelated back to the portion from which they came. Methods ofdesigning sets of bar code sequences is shown for example in U.S. Pat.No. 6,235,475, the contents of which are incorporated by referenceherein in their entirety. In certain embodiments, the bar code sequencesrange from about 5 nucleotides to about 15 nucleotides. In a particularembodiment, the bar code sequences range from about 4 nucleotides toabout 7 nucleotides. In certain embodiments, the bar code sequences areattached to the template nucleic acid molecule, e.g., with an enzyme.The enzyme may be a ligase or a polymerase, as discussed above.Attaching bar code sequences to nucleic acid templates is shown in U.S.Pub. 2008/0081330 and U.S. Pub. 2011/0301042, the content of each ofwhich is incorporated by reference herein in its entirety. Methods fordesigning sets of bar code sequences and other methods for attaching barcode sequences are shown in U.S. Pat. Nos. 6,138,077; 6,352,828;5,636,400; 6,172,214; 6,235,475; 7,393,665; 7,544,473; 5,846,719;5,695,934; 5,604,097; 6,150,516; 7,537,897; 6,172,218; and 5,863,722,the content of each of which is incorporated by reference herein in itsentirety. After any processing steps (e.g., obtaining, isolating,fragmenting, amplification, or barcoding), nucleic acid can besequenced.

Sequencing may be by any method known in the art. DNA sequencingtechniques include classic dideoxy sequencing reactions (Sanger method)using labeled terminators or primers and gel separation in slab orcapillary, sequencing by synthesis using reversibly terminated labelednucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing,allele specific hybridization to a library of labeled oligonucleotideprobes, sequencing by synthesis using allele specific hybridization to alibrary of labeled clones that is followed by ligation, real timemonitoring of the incorporation of labeled nucleotides during apolymerization step, polony sequencing, and SOLiD sequencing. Separatedmolecules may be sequenced by sequential or single extension reactionsusing polymerases or ligases as well as by single or sequentialdifferential hybridizations with libraries of probes.

A sequencing technique that can be used includes, for example, Illuminasequencing. Illumina sequencing is based on the amplification of DNA ona solid surface using fold-back PCR and anchored primers. Genomic DNA isfragmented, and adapters are added to the 5′ and 3′ ends of thefragments. DNA fragments that are attached to the surface of flow cellchannels are extended and bridge amplified. The fragments become doublestranded, and the double stranded molecules are denatured. Multiplecycles of the solid-phase amplification followed by denaturation cancreate several million clusters of approximately 1,000 copies ofsingle-stranded DNA molecules of the same template in each channel ofthe flow cell. Primers, DNA polymerase and four fluorophore-labeled,reversibly terminating nucleotides are used to perform sequentialsequencing. After nucleotide incorporation, a laser is used to excitethe fluorophores, and an image is captured and the identity of the firstbase is recorded. The 3′ terminators and fluorophores from eachincorporated base are removed and the incorporation, detection andidentification steps are repeated. Sequencing according to thistechnology is described in U.S. Pat. Nos. 7,960,120; 7,835,871;7,232,656; 7,598,035; U.S. Pat. Nos. 6,911,345; 6,833,246; 6,828,100;6,306,597; 6,210,891; U.S. Pub. 2011/0009278; U.S. Pub. 2007/0114362;U.S. Pub. 2006/0292611; and U.S. Pub. 2006/0024681, each of which areincorporated by reference in their entirety.

Sequencing generates a plurality of reads. Reads generally includesequences of nucleotide data wherein read length may be associated withsequencing technology. For example, the single-molecule real-time (SMRT)sequencing technology of Pacific Bio produces reads thousands ofbase-pairs in length. For 454 pyrosequencing, read length may be about700 bp in length. In some embodiments, reads are less than about 500bases in length, or less than about 150 bases in length, or less thanabout 90 bases in length. In certain embodiments, reads are betweenabout 80 and about 90 bases, e.g., about 85 bases in length. In someembodiments, these are very short reads, i.e., less than about 50 orabout 30 bases in length.

The sequence reads may be analyzed to characterize the target gene orregion of interest. For example, mutations can be “called” (i.e.,identified and reported), a haplotypte for the sample may be reported,or other analyses may be performed. Mutation calling is described inU.S. Pub. 2013/0268474. In some embodiments, an analysis may includedetermining copy number states of genomic regions of interest. A set ofsequence reads can be analyzed by any suitable method known in the art.For example, in some embodiments, sequence reads are analyzed byhardware or software provided as part of a sequence instrument. In someembodiments, individual sequence reads are reviewed by sight (e.g., on acomputer monitor). A computer program may be written that pulls anobserved genotype from individual reads. In certain embodiments,analyzing the reads includes assembling the sequence reads and thengenotyping the assembled reads.

Sequence assembly can be done by methods known in the art includingreference-based assemblies, de novo assemblies, assembly by alignment,or combination methods. Assembly can include methods described in U.S.Pat. No. 8,209,130 titled Sequence Assembly by Porecca and Kennedy, thecontents of each of which are hereby incorporated by reference in theirentirety for all purposes. In some embodiments, sequence assembly usesthe low coverage sequence assembly software (LOCAS) tool described byKlein, et al., in LOCAS-A low coverage sequence assembly tool forre-sequencing projects, PLoS One 6(8) article 23455 (2011), the contentsof which are hereby incorporated by reference in their entirety.Sequence assembly is described in U.S. Pat. Nos. 8,165,821; 7,809,509;6,223,128; U.S. Pub. 2011/0257889; and U.S. Pub. 2009/0318310, thecontents of each of which are hereby incorporated by reference in theirentirety.

Functions described above such as sequence read analysis or assembly canbe implemented using systems of the invention that include software,hardware, firmware, hardwiring, or combinations of any of these.

FIG. 3 gives a diagram of a system 301 according to embodiments of theinvention. System 301 may include an analysis instrument 303 which maybe, for example, a sequencing instrument (e.g., a HiSeq 2500 or a MiSeqby Illumina). Instrument 303 includes a data acquisition module 305 toobtain results data such as sequence read data. Instrument 303 mayoptionally include or be operably coupled to its own, e.g., dedicated,analysis computer 333 (including an input/output mechanism, one or moreprocessor, and memory). Additionally or alternatively, instrument 303may be operably coupled to a server 313 or computer 349 (e.g., laptop,desktop, or tablet) via a network 309.

Computer 349 includes one or more processors and memory as well as aninput/output mechanism. Where methods of the invention employ aclient/server architecture, steps of methods of the invention may beperformed using the server 313, which includes one or more of processorsand memory, capable of obtaining data, instructions, etc., or providingresults via an interface module or providing results as a file. Theserver 313 may be engaged over the network 309 by the computer 349 orthe terminal 367, or the server 313 may be directly connected to theterminal 367, which can include one or more processors and memory, aswell as an input/output mechanism.

In system 301, each computer preferably includes at least one processorcoupled to a memory and at least one input/output (I/O) mechanism.

A processor will generally include a chip, such as a single core ormulti-core chip, to provide a central processing unit (CPU). A processmay be provided by a chip from Intel or AMD.

Memory can include one or more machine-readable devices on which isstored one or more sets of instructions (e.g., software) which, whenexecuted by the processor(s) of any one of the disclosed computers canaccomplish some or all of the methodologies or functions describedherein. The software may also reside, completely or at least partially,within the main memory and/or within the processor during executionthereof by the computer system. Preferably, each computer includes anon-transitory memory such as a solid state drive, flash drive, diskdrive, hard drive, etc. While the machine-readable devices can in anexemplary embodiment be a single medium, the term “machine-readabledevice” should be taken to include a single medium or multiple media(e.g., a centralized or distributed database, and/or associated cachesand servers) that store the one or more sets of instructions and/ordata. These terms shall also be taken to include any medium or mediathat are capable of storing, encoding, or holding a set of instructionsfor execution by the machine and that cause the machine to perform anyone or more of the methodologies of the present invention. These termsshall accordingly be taken to include, but not be limited to one or moresolid-state memories (e.g., subscriber identity module (SIM) card,secure digital card (SD card), micro SD card, or solid-state drive(SSD)), optical and magnetic media, and/or any other tangible storagemedium or media.

A computer of the invention will generally include one or more I/Odevice such as, for example, one or more of a video display unit (e.g.,a liquid crystal display (LCD) or a cathode ray tube (CRT)), analphanumeric input device (e.g., a keyboard), a cursor control device(e.g., a mouse), a disk drive unit, a signal generation device (e.g., aspeaker), a touchscreen, an accelerometer, a microphone, a cellularradio frequency antenna, and a network interface device, which can be,for example, a network interface card (NIC), Wi-Fi card, or cellularmodem.

Any of the software can be physically located at various positions,including being distributed such that portions of the functions areimplemented at different physical locations.

System 301 or components of system 301 may be used to perform methodsdescribed herein. Instructions for any method step may be stored inmemory and a processor may execute those instructions. System 301 orcomponents of system 301 may be used for the analysis of genomicsequences or sequence reads (e.g., sequence assembly or variantcalling).

In certain embodiments, as part of the analysis and determination ofcopy number states and subsequent identification of copy numbervariation, the sequence read counts for genomic regions of interest arenormalized based on internal controls. In particular, an intra-samplenormalization is performed to control for variable sequencing depthsbetween samples. The sequence read counts for each genomic region ofinterest within a sample will be normalized according to the total readcount across all control references within the sample.

After normalizing read counts for both the genomic regions of interestand control references, copy number states may be determined. In oneembodiment, the normalized values for each sample of interest will becompared to the normalized values for a control sample. A ratio, forexample, may be generated based on the comparison, wherein the ratio isindicative of copy number and further determinative of any copy numbervariation. In the event that the determined copy number of a genomicregion of interest of a particular sample falls within a tolerable level(as determined by ratio between test and control samples), it can bedetermined that genomic region of interest does not present copy numbervariation and thus the patient is at low risk for being a carrier of acondition or disease associated with such. In the event that thedetermined copy number of a genomic region of interest of a particularsample falls outside of a tolerable level, it can be determined thatgenomic region of interest does present copy number variation and thusthe patient is at risk for being a carrier of a condition or diseaseassociated with such.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1: Determination of Copy Number State of SMN1

Approximately 28 samples are collected to determine carrier status withrespect to spinal muscular atrophy (SMA). Genomic DNA is extracted fromwhole human blood using a Gentra Puregene Blood Kit and following thePuregene protocol for DNA Purification from Whole Blood (Qiagen). Of the28 samples, there is 1 water negative control and 7 control DNA samplesand 20 test samples. Each of the control samples includes two or moregenomic regions of interest (e.g. loci) having known (or stable) copynumbers. Control samples 1-4 each include control loci and survivalmotor neuron genes (SMN), including telomeric SMN (SMN1) and centromericSMN (SMN2) genes. There are a total of 17 control loci, 5 SMN1, and 5SMN2, all of which have a known copy number of 2. Control sample 5includes 17 control loci, each having a known copy number of 2, and 5SMN1, each having a known copy number of 0. Control sample 6 includes 17control loci, each having a known copy number of 2, and 5 SMN1, eachhaving a known copy number of 1. Control sample 7 includes 17 controlloci, each having a known copy number of 2, and 5 SMN1, each having aknown copy number of 3 or more. Samples are processed via method 101 toremove copies of SMN2. The sample is treated (e.g., heated) to denaturegenomic dsDNA. Primers specific to SMN2 that are complementary toregions flanking the SMN2 sequence are introduced. The primers areannealed to the ssDNA in the regions flanking the unwanted SMN2 segment.The annealed primers are then extended using a polymerase in atemplate-dependent manner to make double-stranded any single-strandedinstance of SMN2 present in any sample. A double-stranded endonucleaseis introduced and allowed to digest all dsDNA, thus digesting anysegments that include SMN2. This stage of processing of the sample iscompleted by inactivating the ds endonuclease and the remaining DNA isanalyzed for SMN1 by MIP capture and sequencing.

The processed samples are then fragmented and/or denatured inpreparation for hybridization with molecular inversion probes. Thegenomic DNA of each sample is fragmented/denatured by any known methodor technique sufficient to fragment genomic DNA.

Once it is isolated, MIP capture probes are hybridized to the fragmentedgenomic DNA in each sample by introducing capture probe mix into eachsample well. In particular, the capture probe mix will generally includea plurality of SMA molecular inversion probes that are capable ofbinding to one or more of the genomic regions of interest (e.g., SMN1)or the control DNA. A library of molecular inversion probes isgenerated. The library may include a variety of different probeconfigurations. For example, one or more probes are capable ofhybridizing specifically to the control loci and one or more probes arecapable of hybridizing only to SMN1. Of those probes specific to SMN1,some are capable of producing sequences specific to that paralog whilesome are not capable of producing paralog-specific sequences. Thelibrary may also include one or more probes capable of hybridizingnonspecifically to both SMN1 and SMN2. However, since SMN2 segments areremoved from the sample via methods of the invention, copies of SMN2will not interfere with analysis of SMN1.

Diluted probes are introduced to the isolated fragmented genomic DNA ineach sample and the isolated whole genomic DNA is incubated in thediluted probe mix to promote hybridization. The time and temperature forincubation may be based on any known hybridization protocol, sufficientto result in hybridization of the probes to the DNA. After capture ofthe genomic region of interest (e.g., SMN1) the captured region issubjected to an enzymatic gap-filling and ligation step, in accordancewith any known methods or techniques, including those generallydescribed herein. The captured material may further be purified. Thepurified captured DNA is then amplified by any known amplificationmethods or techniques. In one embodiment, the purified captured DNA isamplified using barcode-based PCR. The resulting barcodes PCRs for eachsample are then combined into a master pool and quantified.

After PCR, portions of the PCR reactions for each sample are pooled andpurified, then quantified. In particular, the PCR reactions for allsamples are pooled in equal volumes into one master pool. The mastersample pool is then purified via a PCR cleanup protocol according tomanufacturer's instructions. The purified pool is then run on amicrofluidics-based platform for sizing, quantification and qualitycontrol of DNA, RNA, proteins and cells. In particular, the purifiedpool and control samples (pre-purification) are run on an AgilentBioanalyzer for the detection and quantification of SMN1 probe products.

Next, the sample pool is prepared for sequencing. In a preferredembodiment, Illumina sequencing techniques are used. Prior tosequencing, the sample pool is reduced to 2 nM by diluting with 1× TE.Template DNA for cluster generation is prepared by combining 10micro-Liter of 0.1 N NaOH with 10 micro-Liter of 2 nM DNA library(sample pool) and incubating said mixture at room temperature for 5 min.The mixture is then mixed with 980 micro-Liter of HT1 buffer (Illumina),thereby reducing the denatured library to a concentration of 20 pM. Thismixture is then mixed (e.g., inversion) and pulse centrifuged. Next, 225micro-Liter of the 20 pM library is mixed with 775 micro-Liter of HT1buffer to reduce the library pool to a concentration of 4.5 pM. Thelibrary pool having a concentration of 4.5 pM is used for on-boardclustering in the sequencing.

The sequencing is carried out on the HiSeq 2500/1500 system sold byIllumina, Inc. (San Diego, Calif.). Sequencing is carried out with theTruSeq Rapid PE Cluster Kit and TruSeq Rapid SBS 200 cycle kit(Illumina) and in accordance with manufacturer's instructions. Inaddition to the reagents and mixes included within the kits, additionalreagents are prepared for genomic read sequencing primers and reversebarcode sequencing primers.

The library pool undergoes sequencing under paired-end, dual-index runconditions. Sequencing generates a plurality of reads. Reads generallyinclude sequences of nucleotide data less than about 150 bases inlength, or less than about 90 bases in length. After obtaining sequencereads, they are further processed as described in U.S. Pat. No.8,209,130.

Read counts for a genomic region of interest are normalized with respectto an internal control DNA. Normalized read counts are compared to theinternal control DNA, thereby obtaining a ratio. A copy number state ofthe genomic region of interest is determined based on the comparison,specifically the ratio.

The plurality of reads generated by the sequencing method describedabove are analyzed to determine copy number states, and ultimately copynumber variation, in any of the genomic regions of interest (e.g., SMN1)that would necessarily indicate the presence of an autosomal recessivetrait in which copy number variation is diagnostic (e.g., spinalmuscular atrophy). Analysis of the read counts is carried out usingIllumina's Hi Seq BclConverter software. Files (e.g. qSeq files) may begenerated for both the genomic and barcode reads. In particular, inaccordance with one method of the present invention, genomic read datafor each sample is split based upon the barcode reads, which yieldsseparate FASTQ files for each sample.

Based on the ratios, loci copy numbers may be called as follows: a ratioof <0.1 will be called a copy number state of 0; a ratio between 0.1 and0.8 will be called a copy number state of 1; a ratio between 0.8 and1.25 will be called a copy number state of 2; and a ratio of >1.25 willbe called a copy number state of 3+.

The determined copy numbers can then be used to determine the carrierstatus of an individual from which the sample was obtained (i.e. whetherthe patient is a carrier of the disease). In particular, if the copynumber state is determined to vary from the normal copy state (e.g., CNis 0, 1 or 3+), it is indicative the condition (e.g., carrier of SMA).

What is claimed is:
 1. A method of removing unwanted segments of anucleic acid from a sample, the method comprising: obtaining asingle-stranded nucleic acid that contains an unwanted segment; addingcomplementary nucleic acid to create a double-stranded region thatcontains the unwanted segment; and digesting the double-stranded region,thereby removing the unwanted segment from the nucleic acid.
 2. Themethod of claim 1, wherein adding the complementary nucleic acidcomprises annealing an oligonucleotide to the unwanted segment, therebycreating the double-stranded region.
 3. The method of claim 2, whereinthe annealing step comprises annealing a plurality of primers to aplurality of portions of the nucleic acid that flank an unwantedsegment.
 4. The method of claim 1, wherein adding the complementarynucleic acid comprises: annealing an oligonucleotide to a portion of thesingle-stranded nucleic acid that flanks the unwanted segment; andextending the annealed oligonucleotide to create the double-strandedregion.
 5. The method of claim 4, wherein the annealing step comprisesannealing a plurality of primers to a plurality of portions of thenucleic acid that flank an unwanted segment.
 6. The method of claim 4,wherein the extending step is conducted using a polymerase enzyme underconditions sufficient to cause extension of the primer in atemplate-dependent manner.
 7. The method of claim 1, wherein thedigesting step comprising exposing the sample to an enzyme thatpreferentially digests double-stranded nucleic acid.
 8. The method ofclaim 7, wherein the enzyme is selected from double-strandedendonucleases, restriction endonucleases, and nicking enzymes.
 9. Themethod of claim 8, further comprising the step of deactivating theenzyme.
 10. The method of claim 1, wherein the nucleic acid is selectedfrom DNA, RNA, and modified nucleic acids.
 11. The method of claim 1,further comprising the step of analyzing nucleic acid remaining afterthe digesting step.
 12. The method of claim 1, wherein the digestingstep results in intact genomic DNA lacking one or more unwanted segmentand that is compatible with a nucleic acid analysis assay.
 13. Themethod of claim 12, wherein the assay comprises molecular inversionprobe capture.
 14. The method of claim 13, wherein the assay furthercomprises sequencing.
 15. The method of claim 14, wherein the sequencingis selected from Sanger sequencing and Next Generation Sequencing. 16.The method of claim 1, wherein the unwanted segment is a paralog, apseudogene, or non-paralogous repetitive element.
 17. The method ofclaim 1, further comprising the step of obtaining a sample from asubject and denaturing double-stranded DNA in the sample.
 18. The methodof claim 17, wherein the denaturing step comprises exposing the sampleto heat, a detergent, or a basic solution.